After neurotrauma, neurons in the central nervous system (CNS) do not regenerate their injured axons. Intrinsic signals that progress with normal neuronal differentiation play a significant role in preventing axon regeneration. Several intrinsic negative regulators of regeneration have been identified through studies of knock-out mice. We will design and test novel self-deliverable RNA interference (sdRNAi) compounds as an approach to evaluate intrinsic barriers to regeneration as therapeutic targets. We will use novel sdRNAi technology, which does not require any additional delivery formulation, to reduce the risk of inflammation and non-specific effects, which are of special concern with RNAi injected into the CNS. sdRNA has been proven effective in vivo without the need for special delivery packaging or liposomes. For clinical development, it will be important to determine if sdRNA against intrinsic regulators needs to be injected in the vicinity of the cell body, or if it can be injected at the site of the injured axons. In the latter case, sdRNA might act at the nerve termina or might be retrogradely transported to the cell body. As a focus on delivery techniques, we will investigate sdRNAs applied to cell bodies and axons separately in vitro, and then by injection into the motor cortex or injured spinal cord in vivo. These experiments will help us design appropriate delivery strategies to treat spinal cord injury. We will create sdRNAs against three targets validated through knockout techniques. We will design, synthesize, and test sdRNAs to PTEN, TSC1 and KLF9, all targets shown to promote axon regeneration when silenced. Each candidate will be evaluated in in vitro in primary neurons for knockdown of expression, and for promotion of neurite outgrowth. The effect on astrocyte proliferation will be studied with primary glial cultures. To evaluate delivery, we will test the effect of sdRNA on axon growth in primary cultures plated in compartmental chambers that allow addition of agents specifically to the axonal compartment or to the cell body compartment. The effectiveness of the lead sdRNA will be tested in vivo in a rat contusion model of spinal cord injury. We will evaluate if local or distl application of sdRNA is required to induce gene silencing in vivo and stimulate regeneration of axons. Functional recovery of injured rats will also be assessed to for preclinical validation.